Fig 2: The knockdown of CBX4 attenuates the abilities of proliferation and migration in lung cancer cells via decreasing BMI-1 expression. NCI-H460 and A549 cells were infected with shCBX4 lentivirus or transfected wt-BMI-1 plasmid. A, The expression levels of CBX4 and BMI-1 were measured by Western blotting and analysed by Quantity One software. B, In colony formation assay, cells were fixed and stained with crystal violet after culturing for 2 wk and the stained cells were counted. C, Cells were seeded and stained with crystal violet using transwell assay. Scale bars: 200 µm. (D and E) The relative expression of proteins of proliferation (CDK2, Cyclin E, P53) and migration (MMP2, MMP9, CXCR4) was analysed by Western blotting. GAPDH was used as an internal control. All data were presented as mean ± SD (* P < .05, ** P < .01, *** P < .001)

Fig 3: CBX4 knockdown reduces the proliferation and metastasis of lung cancer in vivo. NCI-H460 and A549 cells were injected into the corresponding part depending on the different models. A, After 5 wk, the nodular tumour was harvested and (B) the volumes of the tumour were measured every other week. The tumours in the CBX4-silencing groups were obviously smaller than in the control group. C, The weights of the xenografts were measured at the end of experiment. D, Images for CBX4, BMI-1 and Ki67 staining are shown for the xenografts. Scale bars: 100 µm. E, The expressions of CBX4 and BMI-1 in xenograft tumours were analysed by Western blotting. F, Representative livers and H&E staining of the metastatic nodules in the livers were from the indicated groups. Metastatic nodules are pointed out by arrows. Statistical results of the metastatic nodules are presented as mean ± SD (* P < .05, ** P < .01, *** P < .001)

Fig 4: The knockdown of CBX4 reduces the migratory ability in NCI-H460 and A549 cells. (A and B) Cells were seeded in 6-well plates and the scratch migration assay was performed to test the migratory potential of NCI-H460 and A549 cells after infecting with shCon or shCBX4 lentivirus. The ratios of the wound closure were measured at three different sites in each image. Scale bars: 200 µm. C, Cells were seeded and stained with crystal violet using transwell assay. D, The histograms show the ratio of the migrated cells to the control from three independent fields. Scale bars: 200 µm. E, Cells were seeded on the coverslips and immunostained with F-actin (green) and DAPI (blue). Scale bars: 10 µm. F, The relative proteins of migration (MMP2, MMP9, CXCR4) and loading control were analysed by Western blot. All data were presented as mean ± SD (*** P < .001)

Fig 5: The relative expression of CBX4 was elevated in human lung cancer tissues and cells. A, Representative immunohistochemical images of CBX4 in human lung cancer and adjacent cancer tissues. Scale bars: 100 µm. B, The CBX4 expression levels in lung cancer and adjacent cancer tissues (n = 30). C, The expression level of CBX4 was analysed by Western blot in lung cancer cells (PC9, NCI-H1299, NCI-H460, A549 and MES-1 cells) and the histograms show the CBX4 relative expression. Densitometric measurements were analysed using Quantity One software. The protein expression levels were normalized to those of the vehicle control (100%). Data are presented as means ± SD (*** P < .001)